The isolation and characterisation of DNA regions which control tissue specific and/or hormonally-regulated gene expression has been an important to the understanding of the developmental processes by which expression of particular genes is limited to specific cell types. Promoter regions are found immediately upstream and often overlapping the start site(s) of transcription and are critical for initiation and basal levels of transcription. Enhancers are regulatory regions which may lie some distance from the transcription start site, either upstream or downstream of a gene or within introns and which often confer high level tissue specific or hormonally-regulated expression; in some cases their action is specific to particular promoters. The function of both promoters and enhancers is mediated by specific proteins, transcription factors, that bind to specific DNA sequences. Alone or in combination with other transcription factors they recruit the core transcription machinery including RNA polymerase to the transcription initiation site and act to stimulate their activity. Isolated promoters and enhancer sequences can be used, in gene therapy for example, to direct expression of other genes in a cell or tissue specific manner and also provide targets for the development of agents that can specifically modulate gene expression.
The promoters and regulatory regions of a number of genes that are expressed in the prostate have been studied either using transfection techniques or by following gene expression in transgenic mice. We have previously compared the cell-type specificity of expression directed by promoters of the prostate-expressed genes, probasin (Pb) and relaxin genes and the promoter and enhancer of the prostate specific antigen (PSA) gene (1). Most of the genes identified as prostate-specific are androgen-inducible and this aspect of their function has been studied in some detail. Thus the importance of androgen response elements for induced expression and/or binding of androgen receptor have been characterised in the PSA (2,3), human glandular kallikrein (KLK2) (4), rat prostatic steroid binding proteins (PSBP) (5,6), probasin Pb (7,8) and prostatic acid phosphatase genes (9) and in regulatory elements in the introns of the rat PSBP C3(1) gene (10) and the rat 20-KDa androgen regulated protein (11).
Among the core promoter regions analysed only that of the probasin gene confers substantial prostate specificity of expression (1,15). Elements involved in conferring prostate-specificity of expression per se, as distinct from androgen responsiveness, have not been well characterised, though tissue-specific factors binding to regions of the PSBP C3 gene promoter and 1st intron have been identified (9,12). The gene for rat PSBP C(3) with 4 kb upstream and 2 kb downstream flanking sequences is expressed tissue-specifically and with appropriate hormonal control in transgenic mice (13). The use of a 5 kb upstream region from the rat PSBP C3(1) gene to express the SV40 T-antigen could elicit prostate tumours, but expression was not highly restricted and other abnormalities were common (14). Studies with transgenic mice have established that regions of the probasin and PSBP C(3) genes can confer prostate specificity.
The PSA and probasin regulatory regions are the two most studied among prostate-expressed genes. It has been established that a 430 bp region upstream of the rat probasin gene is able to confer prostate specificity of expression on reporter genes in transfection experiments (1) and in transgenic animals (15,16); when used to target expression of the SV40 T-antigen, prostate tumours develop specifically (17,18). This expression is not totally specific but specificity is significantly improved by the inclusion of MAR (matrix attachment regions) from the chick lysozyme gene (15). The 430 bp promoter region is strongly responsive to androgen induction and androgen response elements which bind the androgen receptor (AR) have been characterised (4,6,7,16).
The PSA upstream region (to-630 bp) also acts as a strongly androgen responsive promoter and androgen response elements have also been characterised (2,3). However, this region is not sufficient to direct cell type specific expression in culture (1) or tissue specific expression in transgenic mice (19). Use of the 630 bp human PSA promoter region to express an activated Ha-ras oncogene in transgenic mice led to the development of salivary gland and not prostate tumours (19). Pang et al. have reported that the equivalent promoter region isolated from a prostate cancer patient contained 7 mutations compared to the published sequence and was highly active in the prostate cancer cell line LNCaP (20,21). More recently, an enhancer region has been identified in the region 4 to 5 kb upstream of the transcription start site of the PSA gene (20,21). This PSA enhancer has been shown to act as an androgen-inducible enhancer and in combination with the PSA promoter to display significant cell-type specificity (1,20,21).
Prostate-Specific Membrane Antigen
Prostate specific membrane antigen (PSMA) is one of the few prostate-specific proteins identified whose expression is not induced by androgens.
PSMA was first identified as the antigen bound to by the monoclonal antibody 7E11-C5 (25). The antibody was raised against a membrane fraction of the prostate cancer cell line LNCaP and was shown to bind specifically to normal prostate tissue as well as primary and metastatic prostate cancer tissue. This antibody was later found to bind to an internal epitope of this membrane-bound protein (26,27). Subsequently, other monoclonal antibodies targeted to the extracellular domain of the protein have been isolated (28,29).
The cDNA encoding PSMA has been cloned and its sequence determined (30). PSMA is a Type II integral membrane protein and is associated with the plasma membrane of expressing cells such as LNCaP (30). A splice variant of PSMA (Psm′) that lacks the membrane anchor domain and has been shown to be cytoplasmically located has also been identified (31). The ratio of PSMA to Psm has been reported to be increased in prostate cancer as compared with normal prostate or benign hyperplasia (31). PSMA has been shown to possess two related enzymatic activities, it acts as a carboxypeptidase (folate hydrolase) on poly γ-glutamated folates (32) and as a peptidase on the acidic neuropeptide N-acetylaspartyl glutamate (33). This latter activity is consistent with the expression of PSMA or a related protein in the brain.
The specificity of PSMA expression has been studied at both the protein and RNA level. In addition to its major site of expression in the prostate immunohistochemical studies have identified PSMA expression in the duodenum brush border/small intestine, in a subset of proximal tubules in the kidney and in rare cells in the colon (34,35). All other normal issues studies have been negative for expression, except for striated muscle which stains with the 7E11-C5 antibody, but not with antibodies to the external domain of PSMA (28).
Both the 7E11-C5 and external domain antibodies have been found to react with tumour vasculature of a wide range of human tumour types (28,36), indicating specific induction of PSMA expression. PSMA expression has not been identified in any normal vasculature.
RNA expression has been found to largely parallel the protein expression data. RNAse protection analysis identified PSMA mRNA in the prostate, salivary gland and brain and sometimes in the small intestine (37). The identification of PSMA RNA in the brain is consistent with the cloning of a closely related cDNA from rat brain (33). Immunohistochemical analyses have failed, however, to identify antigenically reactive PSMA in human brain tissue.
PSMA expression has been shown to be down regulated in the presence of androgens and expression is generally elevated in late stage prostate cancer and in patients undergoing androgen deprivation or ablation therapies (37,38). Expression of PSMA has also been found to be regulated by a number of growth factors; bFGF, TGF-α and EGF upregulate expression while TNF-α decreases it (39).
The restricted high level expression of PSMA in prostate cells and the induction of its expression in the vasculature of a wide range of tumours make it ideal for the targeting of prostate and other tumour types. Genomic clones encompassing the PSMA gene have been isolated and its sequence and exon/intron structure determined (40). Regulatory regions controlling its expression may find use in gene therapeutic cancer treatments, enabling the restricted or high level expression in the target cell types. Such regulatory regions also provide a target for the development of agents that may interfere with gene expression in the target cell types.